Diagnostic assays for detecting pathogens or other targets have employed labeled DNA probes which are added to DNA samples and permitted to hybridize to the fixed DNA sample. Hybridization indicates the presence of the pathogen or other target. An increase in sensitivity of the assay can be obtained by amplification of the DNA sample.
Relatively recently, a significant improvement in DNA amplification has been provided. Known as the polymerase chain reaction (PCR) technique, it may be generally described in the following manner. The technique is an enzymatic, in vitro synthesis method for replicating or amplifying specific target DNA sequences in DNA samples. The technique employs DNA polymerase, deoxynucleoside triphosphates and two oligonucleotide primers that hybridize to opposite strands of the DNA sample and flank the region of interest in the target DNA sequence. Exponential amplification of the target sequence is obtained by a repetitive series of steps comprising template denaturation, primer annealing and extension of the annealed primers by DNA polymerase, generally referred to as thermal cycling steps. Such a PCR technique is capable of producing amplification of the target sequence, which termini are defined by the 5'-ends of the oligonucleotide primers employed, by a factor of up to about 10.sup.9. The PCR technique is disclosed, for example, in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159 and 4,965,188.
In situ PCR is a relatively new variant of the standard PCR technique. In in situ PCR, the DNA sample is typically a slice of fixed tissue with morphologically recognizable cells. The PCR amplification products remain at, and can be detected at, their site of origin indicating which cells contain the target DNA sequence.
Once a DNA sample is physically amplified in a PCR amplification procedure, detection of the presence or absence of the desired target gene sequence can be accomplished by a variety of isotopic or non-isotopic detection methods. One of the most desirable and preferred methods to detect such PCR target sequence is to amplify the DNA sample in the presence of a labeled molecule, such as a labeled oligonucleotide primer or labeled deoxynucleotide, which becomes incorporated into the amplified DNA. This is generally referred to as direct in situ PCR detection. In general, the labeled molecules are incorporated into nucleotide analogues of dATP, dCTP, dGTP, dTTP or dUTP and are incorporated directly into the PCR amplification products. The labels are generally small molecules like biotin, horseradish peroxidase, digoxigenin, .sup.32 P and the like. PCR amplification products containing these labels can be detected directly, or with enzymes coupled to antibodies to the label. The enzymes can generate any of many different reporter molecules, such as for example, isotopic, fluorescent or colorimetric reporter molecules.
One of the major advantages of direct in situ PCR detection is that many labels can be incorporated into the amplification products at many different sites, particularly if one employs a labeled nucleotide. This generally increases the signal generated and thus lowers the detection limit. A further advantage of direct in situ PCR is that the detection procedure is greatly simplified, since no DNA probes, DNA hybridization or washing steps are required.
Alternatively, unlabeled PCR amplification products can be detected indirectly with a DNA or RNA probe that itself contains a label. This procedure has been variously termed in situ PCR hybridization, indirect PCR detection, or probe-based PCR detection. This method has the disadvantage of requiring several additional steps. Much less signal is also produced, so the detection limit is often adversely impacted. This method, however, provides for better specificity than direct in situ PCR since most nonspecific amplification products will not hybridize to the probe.
Thus, it will be appreciated that there is much to recommend in the direct in situ PCR detection procedure and it would be the procedure of choice in many instances. However, a major limitation to direct in situ PCR detection is that nonspecific PCR amplification products are indistinguishable from specific amplification products. If large amounts of nonspecific products are produced, a false positive may be produced. The production of nonspecific products, even if in reproducible amounts, also raises the background level and therefore negatively impacts the minimum detection level. The quality of results obtained using direct in situ PCR detection is dependent upon the quality of the PCR amplification reaction itself. In contrast, probe-based PCR detection is much less sensitive to the presence of nonspecific products.
Since the discovery of the PCR technique, a number of factors have been identified in solution-phase PCR which impact upon the specificity of the PCR amplification reaction. Among those factors are the following sources of nonspecificity problems: non-optimized PCR protocol, primer nonspecificity, primer oligomerization and primer-independent nonspecificity. The solution to the problem due to non-optimized PCR protocol is to optimize the PCR protocol and thermal cycling parameters. A solution to the problem of primer nonspecificity is the selection of a primer of a different nucleotide sequence. Another solution to the problem of primer nonspecificity, as well as the problem of primer oligomerization, is the so-called hot-start technique disclosed by Chou et al. Nucleic Acid Research, Vol. 20, No. 7 1717-1723 (1992). In this technique, the entire PCR reaction mixture is kept relatively warm at all times once constituted but prior to thermal cycling. This prevents nonspecific primer annealing and extension which could otherwise occur at lower temperatures. The hot-start technique has been demonstrated to be of great value with in situ PCR in lowering nonspecific signal due to primer nonspecificity and primer oligomerization, see Nuovo et al., Amer. Journal of Pathology, Vol. 139, No. 6, 1239-1244 (Dec. 1991).
However, the fourth source of nonspecific signal identified hereinbefore, namely primer-independent nonspecific amplification, is a phenomenon observed in the absence of primers and is not curable by the hot-start technique. This problem of primer-independent nonspecificity has been observed by various investigators who have concluded that the problem is so severe that direct detection of amplified target sequence in situ PCR could not be considered a suitable technique.
While the problem of primer-independent nonspecificity has been illustrated hereinbefore with respect to in situ PCR amplification where it is a particularly bothersome problem, it will be appreciated that such primer-independent nonspecificity can also be a problem in other DNA replication procedures, such a solution-phase PCR amplification or in the primed in situ extension (PRINS) replication technique which uses only a single oligonucleotide primer, which is annealed to chromosomal DNA and extended in situ with the incorporation of labeled oligonucleotides. In the various DNA replication/amplification procedures, the production of primer-independent nonspecific products presents a serious problem in the detection phase of an assay procedure since the nonspecific products can significantly raise the background level to such high levels that false positives are produced and also negatively impact the minimum or lower detection level of the assay.
It is therefore an object of this invention to provide a procedure for reducing or substantially eliminating production of primer-independent nonspecific products during a process for replicating or amplifying and detecting a target DNA sequence in a DNA sample subjected to a DNA replication/amplification process. A further object of this invention is to provide a procedure for reducing or substantially eliminating production of primer-independent nonspecific products during a PCR amplification process, particularly in an in situ PCR amplification process and more particularly in a direct in situ PCR amplification and detection process.